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Genome Biology 2008, 9:R117
Open Access
2008Gaudriaultet al.Volume 9, Issue 7, Article R117
Research
Plastic architecture of bacterial genome revealed by comparative
genomics of Photorhabdus variants
Sophie Gaudriault
*†
, Sylvie Pages
*†
, Anne Lanois
*†
, Christine Laroui
*†
,
Corinne Teyssier
‡
, Estelle Jumas-Bilak
‡
and Alain Givaudan
*†
Addresses:
*
INRA, UMR 1133, Laboratoire EMIP, Place Eugène Bataillon, F-34095 Montpellier, France.
†
Université Montpellier 2, UMR 1133,
Laboratoire EMIP, Place Eugène Bataillon, F-34095 Montpellier, France.
‡
Université Montpellier 1, EA 3755, Laboratoire de Bactériologie-
Virologie, 15, Avenue Charles Flahault, BP 14491, F-34060 Montpellier Cedex 5, France.
Correspondence: Sophie Gaudriault. Email:

© 2008 Gaudriault et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License ( which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: The phenotypic consequences of large genomic architecture modifications within a
clonal bacterial population are rarely evaluated because of the difficulties associated with using
molecular approaches in a mixed population. Bacterial variants frequently arise among Photorhabdus
luminescens, a nematode-symbiotic and insect-pathogenic bacterium. We therefore studied genome
plasticity within Photorhabdus variants.
Results: We used a combination of macrorestriction and DNA microarray experiments to
perform a comparative genomic study of different P. luminescens TT01 variants. Prolonged culturing
of TT01 strain and a genomic variant, collected from the laboratory-maintained symbiotic
nematode, generated bacterial lineages composed of primary and secondary phenotypic variants
and colonial variants. The primary phenotypic variants exhibit several characteristics that are
absent from the secondary forms. We identify substantial plasticity of the genome architecture of
some variants, mediated mainly by deletions in the 'flexible' gene pool of the TT01 reference
genome and also by genomic amplification. We show that the primary or secondary phenotypic
variant status is independent from global genomic architecture and that the bacterial lineages are
genomic lineages. We focused on two unusual genomic changes: a deletion at a new recombination
hotspot composed of long approximate repeats; and a 275 kilobase single block duplication
belonging to a new class of genomic duplications.
Conclusion: Our findings demonstrate that major genomic variations occur in Photorhabdus clonal
populations. The phenotypic consequences of these genomic changes are cryptic. This study
provides insight into the field of bacterial genome architecture and further elucidates the role
played by clonal genomic variation in bacterial genome evolution.
Background
Comparative genomics, in the study of different bacterial gen-
era, species, and strains, leads to the definition of two DNA
pools in bacterial genomes: a set of genes shared by all
Published: 22 July 2008

Genome Biology 2008, 9:R117 (doi:10.1186/gb-2008-9-7-r117)
Received: 15 April 2008
Revised: 12 June 2008
Accepted: 22 July 2008
The electronic version of this article is the complete one and can be
found online at />Genome Biology 2008, 9:R117
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.2
genomes in a taxa, namely the 'core' genome; and a set of
genes containing mobile and accessory genetic elements,
termed the 'flexible' gene pool. Both intergenomic and
intragenomic rearrangements occur in this 'flexible' gene
pool [1]. Changes in the 'flexible' gene pool are considered to
be the motor of bacterial diversification and evolution [2-4].
However, comparative genomic analyses of genomic variants
within a clonal population are rarely undertaken because of
the difficulties involved in using molecular approaches in a
mixed population. Initially, researchers focused on local
modifications of the DNA sequence occurring during phase
variation. Phase variation is an adaptive process by which cer-
tain bacteria within a bacterial subpopulation, called phase
variants, undergo frequent and reversible phenotypic
changes. Phase variation is dependent on DNA sequence
plasticity, generating a reversible switch between 'on' and 'off'
phases of expression for one or more protein-encoding genes.
Variation in the expression of certain genes in some phase
variants allows the bacterial population to adapt to environ-
mental change [5-7]. Other studies have focused on DNA
sequence variations that involve large regions of the genome
in a clonal population. These extensively distributed and
large genomic rearrangements mostly occur through homol-

ogous recombination between repeated sequences such as
rrn loci, duplicated genes, or insertion sequences, which may
then lead to the inversion, amplification, or deletion of chro-
mosomal fragments. These events can occur either under
strong selective pressure - such as in vitro antibiotic selection
[8], stressful high temperature [9], long-term storage [10-12],
and chronic clinical carriage [13] - or without specific selec-
tive pressure [14-20].
The phenotypic consequences of such large rearrangements
are variable. In Streptomyces spp., genetic instability affects
various phenotypical properties, including morphological dif-
ferentiation, production of secondary metabolites, antibiotic
resistance, secretion of extracellular enzymes, and gene
expression for primary metabolism, regardless of selective
pressure [20]. In other bacterial species and when stressful
selective pressure is applied, large-scale genomic variation
often correlates with modification of certain phenotypes:
reversion from nutritional auxotrophy to prototrophy [10],
variation in colony morphology [11], modification of bacterial
growth features [12], and adaptation to high temperature [9].
Few data are available on phenotypic variation in the absence
of strong selective pressure. A few studies suggest that large
genomic architecture modifications can occur with or without
slight detectable phenotypic modifications [15,16]. We stud-
ied genomic rearrangements in the entomopathogenic bacte-
rium Photorhabdus luminescens, for which variants are
frequently observed in standard growth conditions, in order
to investigate further the link between genomic variation
within a bacterial population and the phenotypic
consequences.

P. luminescens is a member of the Enterobacteriaceae; it is a
symbiont of entomopathogenic nematodes and is pathogenic
for a wide variety of insects [21-24]. Bacterial variants fre-
quently arise within the Photorhabdus genus. Two types of
variant exist. The phenotypic variants (PVs) are the most
studied. The primary PV is characterized by the presence of
numerous phenotypic traits (production of extracellular
enzymes, pigments, antibiotics, crystalline inclusion bodies,
and ability to generate bioluminescence) that are absent from
the secondary PV. Secondary PVs are mostly obtained during
prolonged in vitro culturing [25,26]. Only primary PVs sup-
port nematode growth and development both in the insect
cadaver and in vitro. However, both variants are equally vir-
ulent to insect hosts [27]. This phenomenon differs from clas-
sical phase variation because it occurs at low and
unpredictable frequency, it is rarely reversible, and numerous
phenotypic traits are altered simultaneously [27]. Recent
studies suggest that generation of PVs in P. luminescens may
be controlled by several regulatory cascades, each of them
involving the products of many different genes [28-31].
The other common variants in Photorhabdus are colonial
variants (CVs). Different colonial morphotypes can be gener-
ated from one colony subculture. This variation is unstable;
indeed, each morphotype can generate all other morphotypes
[32-36]. The most frequent CVs are small-colony variants
(SCVs). These SCVs constitute a slow-growing bacterial sub-
population with atypical colony morphology and unusual bio-
chemical characteristics that, in the case of clinical isolates,
cause latent or recurrent infections [37]. In Photorhabdus,
these SCVs can be generated from primary or secondary PV

[34]. SCVs have small cells, do not produce crystalline inclu-
sions [32-34], and have undergone changes in their proteome
[33,34]. Some SCVs have modified virulence properties and
do not support nematode development and reproduction
[32].
Previous studies, incorporating local genetic [28,38,39] or
nonexhaustive genomic comparisons [33,34,40,41], have not
identified genomic differences within sets of PVs or CVs. We
used the recently elucidated complete nucleotide sequence of
the P. luminescens subspecies laumondii strain TT01 [42] to
study systematically the link between phenotypic and
genomic variations in clonal Photorhabdus variants. We
undertook whole-genome comparisons between the wild-
type TT01 strain and six different PVs or CVs. We showed that
large genomic rearrangements occurred in vivo and in vitro.
We described two categories of intragenomic rearrange-
ments: deletion events occurring in the 'flexible gene pool',
and an unusual duplication of a 275-kilobase (kb) region,
encompassing 4.8% of the TT01 wild-type genome. These
rearrangements were not correlated with the generation of
PVs, and we did not detect a functional relationship between
the genes affected by rearrangements and phenotypic varia-
tion. Thus, the consequences of these genomic changes are
cryptic.
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.3
Genome Biology 2008, 9:R117
Results
TT01α
/I
: a genomic variant isolated from the

laboratory-maintained nematode Heterorhabditis
bacteriophora
The nematode Heterorhabditis bacteriophora TH01, harbor-
ing the TT01 wild-type strain, was collected in Trinidad in
1993 [43]. The nematode was maintained in the laboratory
and multiplied by infestation in the Lepidopteran Galleria
mellonella [44]. In 1998, a further bacterial isolate was taken
from this nematode. During the course of a genetic study of
the type III secretion system, we discovered that the bacte-
rium isolated in 1998 is a genomic variant. It differs from the
TT01 wild-type strain by a 250 base pair deletion at the 5' end
of the gene lopT1 (Additional data file 1). This gene encodes a
type III secretion system effector that appears to be involved
in the depression of the insect innate immune system [45].
Both TT01 wild-type and the lopT1 genomic variant produced
many of the phenotypes associated with primary PVs, includ-
ing bioluminescence, lipase activity, antibiotic production,
and presence of cytoplasmic crystal (Table 1). Therefore, both
were primary PVs. To distinguish between them, the TT01
wild-type strain was named TT01
/I
and the lopT1 genomic
variant, TT01α
/I
(Figure 1).
Isolation and characterization of PVs and CVs from
TT01
/I
and TT01α
/I

We cultured TT01
/I
and TT01α
/I
in liquid broth and selected
primary and secondary PVs on NBTA (nutrient agar supple-
mented with bromothymol blue and triphenyl 2,3,5 tetrazo-
lium chloride) plates. TT01
/II
secondary PV was derived from
TT01
/I
(TT01 lineage; Figure 1). TT01α
/II
and TT01α'
/II
sec-
ondary PVs were obtained from TT01α
/I
(TT01α lineage; Fig-
ure 1). TT01
/II
, TT01α
/II
, and TT01α'
/II
had classic secondary
PV traits (Table 1).
We developed a new agar medium, the TreGNO (nutrient
agar with trehalose and and bromothymol blue) medium, for

color discrimination of TT01 PVs (see Materials and methods
[below] for details). PVs produce green, convex, and mucoid
colonies whereas secondary PVs produce yellow, flat, and
nonmucoid colonies on this medium. TT01
/II
and TT01α
/II
colonies were homogeneous and had the colonial traits of sec-
ondary PVs. However, TT01α'
/II
was composed of three CVs
(TT01α' lineage; Figure 1). The first was a primary colonial
form (green, convex, and mucoid colonies), named REV
because it resembled a revertant colony, exhibiting primary
PV traits (although bioluminescence, pigmentation, and crys-
tal production were not completely restored; Table 1). The
second was a secondary colonial form (yellow, flat, and non-
mucoid colonies), named VAR because of its secondary PV
traits (Table 1). The third form had small, green, convex, and
mucoid colonies, and was named INT because of its interme-
diate traits or traits from both the primary and secondary PVs
(Table 1). These CVs are unstable because each individual
TT01α'
/II
colony grown in liquid broth gives rise to a mixture
of the three colonial forms on TreGNO medium. We gener-
ated a stable secondary PV from the VAR colonial variant by
plating a liquid subculture from an individual VAR colony on
nutrient agar and picking another VAR colony for a new cycle
of liquid/plate culture. We continued this enrichment process

until the liquid subculture generated 95% of VAR colonies on
TreGNO plates. The stable population was named VAR* (Fig-
ure 1).
We PCR-amplified the lopT1 5' region from TT01
/II
, TT01α
/II
,
TT01α'
/II
, VAR*, and REV (Additional data file 1). The lopT1
deletion was only present in the TT01α and TT01α' lineages.
Virulence of TT01 variants
We injected TT01
/I
, TT01
/II
, TT01α
/I
, TT01α
/II
, and VAR* into
Table 1
Phenotypes of P. luminescens TT01
/I
, TT01α
/I
, and their respective variants
Phenotype TT01
/I

TT01α
/I
TT01
/II
TT01α
/II
TT01α'
/II
VAR VAR* REV INT
Bioluminescence + + - - - - - +/w w
Colony morphology Convex,
mucoid,
Convex,
mucoid,
Flat,
nonmucoid
Flat,
nonmucoid
Flat,
nonmucoid
Flat,
nonmucoid
Flat,
nonmucoid
Convex,
mucoid
Small, convex,
mucoid
Lipase activity on
Tween 20-60

++ ++ + + + + + ++ ND
Lipase activity on
Tween 80-85
++ ++ +/w +/w +v + v ++ ND
Pigmentation +(Orange) +(Orange) +(Yellow) ++(Yellow) - - - +(Orange) ND
Antibiotic
production
++- - - - - +/wND
Crystal proteins + + - - - - - w -
Coloration on
TreGNO medium
Green Green Yellow Yellow Yellow Yellow Yellow Green Green
+, positive; -, negative; v, variable; w, weak.
Genome Biology 2008, 9:R117
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.4
Spodoptera littoralis larvae to evaluate the pathogenicity of
these variants in insect larvae. TT01
/II
, TT01α
/I
, and TT01α
/II
had the same level of pathogenicity as TT01
/I
; 50% mortality
(LT
50
) was reached between 28 and 32 hours after injection
for the TT01 wild-type strain and these three variants. By con-
trast, VAR* had a delayed LT

50
of 53 hours, although 100%
mortality was reached at 3 days after infection (Figure 2).
Extensive rearrangements in genomic architecture
correlated with the variant lineages
We examined the whole genome architecture of each variant
using I-CeuI genomic macrorestriction and pulsed field gel
electrophoresis (PFGE) in order to detect large rearrange-
ment such as deletions and amplifications by recombination
between rrn or deletions, amplifications, and translocations
inside I-CeuI fragments. I-CeuI is an intron-encoded enzyme
that specifically cleaves a 26-base-pair site in the bacterial
23S rRNA gene. The PFGE pattern obtained for the TT01
/I
strain matched the pattern of I-CeuI fragments predicted
from the complete TT01
/I
genome sequence (Figure 3a, b;
also see Additional data file 2 for the details of the gels). Using
the TT01
/I
pattern used as a reference, we observed large
genomic rearrangements in TT01α
/I
, TT01α
/II
, TT01α'
II
,
VAR*, and REV. PFGE patterns revealed identical profiles for

primary and secondary PVs within both TT01 and TT01α lin-
eages (Figure 3b and Additional data file 2). Therefore, PV
status (primary versus secondary) in these variant lineages is
independent from global genomic architecture.
Cluster analysis of the seven observed I-CeuI patterns reveals
that variant lineages are in fact genomic lineages (Figure 3c).
The TT01 and TT01α lineages exhibit genomic homogeneity.
The TT01α' lineage shared common genomic features with
the TT01α lineage, but exhibited a more polymorphic
genomic pattern than TT01 and TT01α lineages.
The PFGE patterns of TT01α and the TT01α' lineages only
reveal six apparent I-CeuI fragments, instead of seven frag-
ments in the TT01
/I
reference chromosome; however, the
intensity of the 295-kb band suggests that it may represent
two different fragments. We used Southern blot analysis to
confirm that the seven rrn copies are present in all the vari-
ants (Additional data file 3). Therefore, variation in I-CeuI
PFGE patterns among the TT01 variants appeared to be
unrelated to deletion or amplifications mediated by recombi-
nation between rrn operons.
Additionally, the 465 kb faint band in the TT01α'
/II
pattern
(white star in Additional data file 2) corresponded to a frag-
ment in the REV pattern, suggesting the existence of a 'REV-
like' chromosome subpopulation in TT01α'
/II
.

Deletions and amplifications in the TT01α
/I
and VAR*
variants, representative of the TT01α and TT01α'
lineages
Large genomic rearrangements were present in the TT01α
and TT01α' lineages. We further evaluated the nature of these
rearrangements by comparing gene content between
representative variants of each lineage, TT01
/I
, TT01α
/I
and
VAR*, using genomic DNA hybridization on a P. luminescens
TT01
/I
microarray.
Totals of 159 and 162 genes were absent from TT01α
/I
and
VAR*, respectively (see Additional data file 4). We located
these genes on a circular map of the TT01
/I
chromosome (Fig-
Schematic representation of TT01 variants selection on TreGNO mediumFigure 1
Schematic representation of TT01 variants selection on TreGNO medium.
TT01
/I
, TT01α
/I

, and REV colonies are green, convex, and mucoid
colonies; TT01
/II
, TT01α
/II
, VAR, and VAR* colonies are yellow, flat, and
nonmucoid; and the INT colonies are small, green, convex, and mucoid.
* TT01
/ I
* TT01
/ I
TT01
/ II
TT01
/ II
TT01
/ II
Phenotypic
variation
Phenotypic
variation
=
Genomic
variation
VAR*
INT
VAR
REV
Stabilization
Variant

lineage
TT01
TT01
TT01
* isolated from nematodes
Mortality in Spodoptera littoralisFigure 2
Mortality in Spodoptera littoralis. Shown is the mortality in S. littoralis
infected with the TT01
/I
Photorhabdus luminescens wild-type strain, the
genomic variant TT01α
/I
, the secondary variants TT01
/II
and TT01α
/II
, and
the stabilized VAR* colonial variant. Bacteria obtained at the end of the
exponential phase were injected into fourth-instar larvae. Mortality values
are based on data obtained after injection into 20 larvae. All experiments
were repeated at least twice.
0
50
100
0 1020304050607080
% Mortality
TT01
TT01 /2
TT01_
TT01_

/2.1
TT01_
/2.2 VAR*
TT01
/ I

TT01
/ II
TT01
/ II
VAR*
TT01
/ I
Hours after injection
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.5
Genome Biology 2008, 9:R117
ure 4); they mostly clustered into eight regions absent from
both the TT01α
/I
and VAR* genomes (regions A, C, D, E, F, G,
I, and J) and one region specifically absent from the VAR*
genome (region H). The deleted regions were located
throughout the chromosome, with no particular symmetry
around the replication origin or termination site. Several
regions displayed a GC bias inversion (C, D, E, G, I, and J).
Three overlapped with phagic regions (C, G, and I), suggest-
ing that prophage excision occurred during the TT01
/I
to
TT01α

/I
transition (Table 2). As well as phagic genes, the
deleted regions encompass putative mobile and recombina-
tion-mediating elements such as insertion sequences and
recombination hotspot (Rhs) elements (region A), and plas-
mid-related protein-encoding genes (region J)(Table 2). The
regions C, D, E, and F potentially encode peptide synthetases
involved in antimicrobial compound synthesis (Table 2).
However, we did not observe any significant difference in
antimicrobial activity between TT01
/I
and TT01α
/I
tested for
14 indicator strains (data not shown).
A more thorough analysis of hybridization ratios revealed
that 122 genes had a ratio higher than 1.4 in the VAR* genome
(Additional data file 5). In contrast, comparison of the TT01
/I
and TT01α
/I
genomes revealed only four genes with a ratio
higher than 1.4. These findings suggest that numerous genes
are amplified in the VAR* genome. Among these potentially
amplified genes, 112 are clustered in a unique and large 275-
kb region, named B. This region encompasses 4.8% of the
TT01
/I
genome (from plu0769 = mrfA to plu0980 = hpaA;
Figure 4). Region B is located within the first quarter of the

TT01
/I
chromosome and is not delimited by obvious repeat
elements. According to TT01
/I
genome annotations, the
region B may be involved in numerous and different functions
(Table 2): basal cellular functions involving the DNA
polymerase III ε chain (plu0943 = dnaQ), enolase (plu0913 =
eno), and proteins involved in tryptophan metabolism
(plu0799 = tnaA; plu0800 = mtr); and environment and/or
host interactions, involving the major fimbrial biosynthesis
locus (plu0769-0778 = the mrfABCDEFGHJ operon), insec-
ticidal toxin proteins (plu0805 = tccA3; plu0806 = tccB3;
plu0960 = tcc2; plu0961 = tcdB1; plu0962 = tcdA1; plu0964
= tccC5; plu0965 = tcdA4; plu0970 = tcdB2; plu0971 =
tcdA2), and proteins similar to pyocins (plu0884; plu0886-
0888; plu0892; plu0894).
To determine whether DNA microarray experiments explain
the architectural modifications observed by macrorestriction
experiments, we compared the two sets of data. The observed
I-CeuI macrorestriction fragments from the TT01α lineage
(36 kb, 295 kb, 295 kb, 330 kb, 465 kb, 610 kb, ~3600 kb)
were similar to the theoretical I-CeuI fragments calculated
after size subtraction of the eight deleted regions from the
TT01
/I
I-CeuI fragments (36 kb, 244 kb, 266 kb, 330 kb, 462
kb, 627 kb, ~3478 kb). Therefore, large-scale deletion events
appear to underlie the TT01 to TT01α lineage transition. DNA

microarray experiments in the TT01α' lineage identified a 275
kb amplification of the TT01
/I
genome. Duplication or tripli-
cation of region B may account for the increase in genome size
Variation in genomic architecture of the TT01 variantsFigure 3
Variation in genomic architecture of the TT01 variants. (a) Schematic representation of the I-CeuI restriction map of the TT01
/I
Photorhabdus luminescens
reference genome. (b) Schematic reconstruction of I-CeuI pulsed field gel electrophoresis (PFGE) patterns for TT01
/I
and the six variants representing gels
presented in Additional data file 2. Fragment sizes were calculated using the TT01
/I
genome as a reference. Lane 1: TT01
/I
. Lane 2: TT01
/II
. Lane 3: TT01α
/
I
. Lane 4: TT01α
/II
. Lane 5: TT01α'
/II
. Lane 6: VAR*. Lane 7: REV. (c) Clustering of the PFGE patterns. Patterns were compared using the Dice coefficient
for each pair. Patterns were clustered by UPGMA.
(a)
TT01
/ I

chromosome
5,7 Mbases
478
kb
266
kb
671 kb
3664
kb
330 kb
244
kb
36
kb
(c)(b)
M2
M1
244 kb -
36 kb -
266 kb -
330 kb -
478 kb -
671 kb -
3600 kb -
1345726
5
S000000002
S000000001
S000000003
S000000004

S000000007
S000000005
S000000006
5
TT01
/ I

TT01
/ II
TT01
/ I
TT01

/ II
VAR *
REV
TT01
/ II
Genome Biology 2008, 9:R117
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.6
(~100 kb to 650 kb) observed by macrorestriction for the
TT01α to TT01α' transition. Therefore, duplication appears to
be mainly responsible for the TT01α to TT01α' lineage
transition.
Homologous recombination between long repeats led
to serial deletions of the region H in the TT01α and
TT01α' lineages
We first examined the genomic deletions observed in the
TT01α
/I

and VAR* variants. We focused on region H, which,
by contrast to other deleted regions, did not exhibit typical
recombination-mediating elements. Probes targeting differ-
ent parts of the region H were hybridized on genomic DNA of
the wild-type strain and the six variants. Hybridization pat-
terns were identical within each variant lineage and con-
firmed the presence of a 25 kb deletion within the region H
(from plu3237 to plu3253) for the TT01α' lineage (data not
shown). Southern analysis also indicated the presence of a
small deletion of about 10 kb (from plu3238 to plu3248) in
the TT01α lineage. To map the deletion borders accurately,
primers flanking the 25-kb deletion (R-3236 and F-3254) and
the 10-kb deletion (R-3238bis and F-3249) were designed
(Figure 5) and used for PCR amplification in the TT01α' and
TT01α lineages. Amplified fragments of 4.8 kb and 5.2 kb
were observed (data not shown). These fragments were
sequenced for TT01α
/I
and VAR*, and the deletion was phys-
ically mapped (a genetic map of the region H is presented in
Figure 5). The deletions in TT01α
/I
and VAR* were 12,820
bases (from coordinates 3,833,904 to 3,846,723) and 25,140
bases long (from coordinates 3,830,001 to 3,855,140),
respectively.
We used Nosferatu, software that can detect approximate
repeats in large DNA sequences [46]. The region H is rich in
pairs of repetition units (RPT) larger than 1 kb (Figure 5).
Each deletion began at the right-hand extremity of the first

repetition and finished at the right-hand extremity of the cor-
responding second repetition (RPT179385 repetitions for the
10-kb deletion and RPT179383 repetitions for the 25-kb dele-
tion). Therefore, successive deletions mediated by homolo-
gous recombination between RPT are likely to have occurred
in the region H during the TT01
/I
to TT01α
/I
to VAR* transi-
tion, leading to genomic reduction.
A single block duplication of region B is specific to the
TT01α' lineage
In a second set of analyses, we focused on the gene amplifica-
tion observed in region B, occurring in the TT01α
/I
to VAR
transition. Quantitative PCR was performed for two genes in
region B, mrfA (plu0769) and dnaQ (plu0943). Comparison
of VAR and TT01α
/I
data confirmed that these two genes were
duplicated in the VAR* genome (Figure 6a).
In order to determine whether region B is duplicated specifi-
cally in the VAR* variant or in all variants of the TT01α'
lineage, a probe covering the entire region B (the probe B) was
prepared and hybridized to genomic DNA of the wild-type
strain and the six variants. According to the TT01
/I
genome

sequence, NotI hydrolysis generates 25 fragments with a
unique 1,056-kb fragment containing region B. Hybridization
of the probe B to NotI-hydrolyzed genomic DNA generated a
unique fragment of 1,056 kb in the TT01 lineage and of 1,020
kb in the TT01α lineages (Figure 6b, c). By contrast, in the
Table 2
Deleted and amplified regions in the TY01α
/I
and VAR* genomes
Locus Probable nature
of event
Gene region Size (in kb) Products of interest (similarity or function) Matching GI
a
or EVR
b
A Deletion plu0338-plu0355 18 DNA cytosine, ethyl-transferase, mismatch
repair endonuclease, unknown proteins, Rhs
proteins, IS630 family
Part of GI plu0310-plu0373
B Amplification plu0769-plu0980 275 Proteins involved in basal metabolism (DNA
polymerase III ε chain, enolase, tryptophan
metabolism) and in interaction with
environment and/or host (fimbrial biosynthesis,
Tc insecticidal toxins, pyocins)
Encompassed GI plu0884-plu0901, GI
plu0914-plu0938, and overlapped a
part of GI plu0958-plu1166
C Deletion plu1086-plu1123 44 Unknown proteins, phage regulators, peptide
synthetase, transposase, bacteriophage
proteins

Part of GI plu0958-plu1166
D Deletion plu1861-plu1876 12 Antibiotic biosynthesis Part of GI plu1859-plu1894
E Deletion plu2191-plu2200 11 Antibiotic synthesis and transport Part of EVR plu2179-plu2224
F Deletion plu2468-plu2476 8 unknown protein, ABC transporter, toxoflavin
biosynthesis, transposase
EVR plu2468-plu2476
G Deletion plu2874-plu2960 54 Bacteriophage proteins Part of GI plu2873-plu3038
H Deletion plu3238-plu3252 22 Unknown proteins, VgrG proteins Part of GI plu3207-plu3275
I Deletion plu3380-plu3504 89 Bacteriophage proteins Part of GI plu3379-plu3538
J Deletion plu4324-plu4328 12 Unknown and plasmid-related proteins EVR plu4319-plu4332
a
Genomic islands described in [42].
b
Enterobacterial variable regions described in [56].
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.7
Genome Biology 2008, 9:R117
TT01α' lineage, the B probe hybridized to the 1,020-kb frag-
ment and an additional fragment. This second fragment has a
similar size in TT01α'
/II
and VAR* variants (610 kb) but is
smaller (365 kb) in the REV variant. These findings showed
that duplication of region B occurred in all TT01α' lineage
variants.
Region B encompasses 275 kb in the TT01
/I
genome
sequence; thus, we determined whether the resulting ampli-
fied genes were dispersed in the genome or co-localized in an
unique block. The unique additional fragment detected by the

probe B in the TT01α'
/II
and VAR* variants indicated that the
product of the region B amplification is constituted either of
one block or a few blocks co-localized in a genomic region
whose size is smaller than 610 kb in TT01α'
/II
and VAR* and
smaller than 365 kb in REV. The probe B was also hybridized
to ApaI-hydrolyzed genomic DNA of the wild-type strain and
the six variants. The seven patterns were identical and the
probe B hybridized with the two main 74 and 156 kb frag-
ments covering the major part of region B according to the
TT01
/I
genome reference sequence (data not shown). Because
the duplication did not modify the ApaI restriction pattern,
we concluded that region B was amplified as a single block.
Schematic representation of DNA microarray data as a circular map of the TT01
/I
genomeFigure 4
Schematic representation of DNA microarray data as a circular map of the TT01
/I
genome. Circle 1 (from outside to inside): scale marked in megabases.
Circle 2: location of transposases (red) and phage-related genes (green) location. Circles 3, 4, and 5: DNA microarray data comparing TT01
/I
and TT01α
/I
genomes (circle 3), TT01
/I

and VAR* genomes (circle 5), and synthesis from both experiments (circle 4). Deleted genes are represented by bars inside the
circle. Amplified genes are represented by bars outside the circle. Deleted and amplified regions are circled in blue. Circle 6: GC bias (G-C/G+C). Circle
7: GC content with <32% G+C in light yellow, between 32% and 53.6% G+C in yellow, and with >53.6% G+C in dark yellow.
5
A
0
1
2
4
3
C
D
E
F
G
I
J
H
B
TT01
/ I
versus TT01
/ I
TT01
/ I
versus VAR*
TT01
/ I
versus VAR*
Genome Biology 2008, 9:R117

Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.8
Discussion
Variant lineages are genomic lineages characterized by
extensive genomic rearrangements
Our study provides the first extensive investigation into
genomic rearrangements in Photorhabdus variants. First, we
evaluated phenotypic traits of the three variant lineages (Fig-
ure 1). The TT01 lineage is derived from the TT01
/I
strain,
which was isolated from the H. bacteriophora TH01 nema-
tode collected in Trinidad in 1993 [43] and whose genome is
sequenced [42]. The TT01α lineage is derived from the
TT01α
/I
genomic variant, which was collected from H. bacte-
riophora TH01 maintained and multiplied in the laboratory.
The TT01α' lineage was derived from the TT01α
/I
variant
after prolonged culture in synthetic medium. Each lineage is
composed of PVs, whereby the primary form is characterized
by the presence of typical phenotypic traits that are absent
from the secondary form. The TT01α' lineage has an addi-
tional level of complexity, because the PVs exhibit features of
CVs such as unstable morphotypes.
We then examined the genomic architecture of each variant
in macrorestriction experiments and used comparative DNA
microarray hybridization experiments to analyze the genomic
content of representative variants for each lineage. Our

findings revealed that large genomic rearrangements charac-
terize each variant lineage. Consequently, these findings pro-
vide insight into probable scenarios underlying each lineage
transition. The whole-genome organization of the TT01 line-
age is described by the TT01 reference genome [42]. Large-
scale deletion events in the TT01 flexible gene pool seem to be
involved in the TT01 to TT01α lineage transition. Deletion
events in the TT01 flexible gene pool and a single block dupli-
cation encompassing 4.8% of the TT01 reference genome
appear to underlie the TT01 to TT01α' lineage transition. The
genomic clusters do not depend on the PV status (see below).
Thus, each variant lineage is a genetic lineage.
Successive deletions between homologous repeats in the region HFigure 5
Successive deletions between homologous repeats in the region H. Genetic map of TT01
/I
region H is shown (blue boxes are open reading frames [ORFs]).
Location of repetition units (RPT) larger than 1 kilobase (kb) is indicated (hatched colored boxes). RPT were systematically searched on the whole TT01
/I
genome sequence by using Nosferatu, software that can detect approximate repeat sequences [46]. The RPTs are numbered according their position on
the chromosome. DNA microarray data for the TT01α
/I
and VAR* genomes are indicated. '+': the gene is present. '-': the gene is absent. '?': the gene is not
represented on the microarray. Schematic representation of TT01α
/I
and the VAR* variant deletions is shown. Deletion borders were obtained from
sequencing between the R-3236 and F-3254 primers in the VAR* variant, and between the R-3238bis and F-3249 primers in the TT01α
/I
variant. Green and
hatched gray boxes represent regions in TT01α
/I

and VAR* genomes variants that were found to be present or absent, respectively. The deleted regions
encompassed sequence between coordinates 3.833.904 and 3,846,724 in TT01α
/I
genome and coordinates 3,830,001 and 3,855,141 in VAR* genome.
RPT179359 RPT179359
RPT179379 RPT179379
RPT179373 RPT179373
RPT179381 RPT179381
RPT179383 RPT179383
1000 bp
RPT179385 RPT179385
RPT179405 RPT179405
R-3236 R-3238bis
F-3249
F-3254
TT01
/ I
plu3226
plu3227
plu3228
plu3229
plu3231
plu3232
plu3235
plu3236
plu3237
plu3238
plu3239
plu3241
plu3242

plu3243
plu3244
plu3245
plu3246
plu3247
plu3248
plu3250
plu3251
plu3252
plu3253
TT01
/ I
+???
+
??
??
++ ? + +
+
????- + ? ?++++ ?+
microarray
data
TT01
/ I

sequence data
3833903
VAR*
+???
+
??

??
++ ? - -
+
????- - ? ? ?+
microarray
data
VAR* sequence data
3830000
plu3254
3846724
3855141
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.9
Genome Biology 2008, 9:R117
Deletion at new recombination hotspot
To explain the molecular mechanisms involved in the rear-
rangements in our variants, we investigated potential
repetitive elements and recombination-mediating elements
flanking the rearranged regions. Large genomic architectural
changes are often driven by homologous recombination
between repeated sequences. The nature of the change then
depends on the relative orientation, size, and spacing of the
repeated sequences [47-50]. Recombination events often
occur at the rrn operon in Gram-negative bacteria, such as
Salmonella, Rhizobium, Escherichia coli, and Ochrobactrum
[11,13,18,51,52]. However, despite the variation detected in
PFGE analysis of the rrn skeleton for the three variant
lineages, we demonstrated that the rearrangements are not
the result of rrn recombination.
Apart from homologous recombination, rearrangements can
be induced by site-specific recombination, associated with

recombination-mediating elements such as mobile elements,
or by illegitimate recombination, linked to shortly spaced
repeats [49,50]. Most of the deleted regions in the TT01α lin-
eage are rich in potential rearrangement-mediating elements,
with both repeated sequences - including insertion sequences
and Rhs elements - and mobile elements, including phagic
and plasmid-related genes.
Genomic annotation of the region H, which underwent suc-
cessive deletions in the TT01α
/I
and VAR* variants, did not
describe the presence of typical repetitive or recombination-
mediating elements. The region H belongs to a large genomic
island containing the genes vgr and hcp, initially described as
genes associated with Rhs elements. Rhs elements are
repeated sequences in the E. coli genome that mediate major
Duplication of region BFigure 6
Duplication of region B. (a) Quantitative PCR was carried out for mrfA (plu0769) and dnaQ (plu0943) using genomic DNA from TT01α
/I
and VAR* variants
and specific internal primers for each gene. pilN (plu1051) and fliC (plu1954) were used for negative controls. PCR was performed in triplicate and data are
presented as ratios, with gyrB as the control gene (95% confidence limits). (b, c) Pulsed field gel electrophoresis (PFGE) of NotI-hydrolyzed genomic DNA
from TT01α
/I
and the six variants following by Southern blot and hybridization with a probe covering the region B (probe B). The PFGE conditions allow
separation of NotI fragments between 50 and 400 kb (panel b) or between 350 and 1,350 kb (panel c). Gray arrows indicate fragments that hybridize with
the probe B. Lane 1: TT01
/I
. Lane 2: TT01
/II

. Lane 3: TT01α
/I
. Lane 4: TT01α
/II
. Lane 5: TT01α'
/II
. Lane 6: VAR*. Lane 7: REV.
(c)(b)
50 kb -
200 kb -
150 kb -
100 kb -
300 kb -
400 kb -
1345 726
(a)
0
0,5
1
1,5
2
2,5
3
pilN fliC mrfJ dnaQ
copy number of the gene/copy
number of gyrB
TT01a
/I
VAR*
TT01

/ I
VAR*
1000 kb -
1345 726
1345726
1345 726
1020 kb
1056 kb
610 kb
365 kb
#600/1000kb
#400 kb
Genome Biology 2008, 9:R117
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.10
chromosomal rearrangements [51,53,54]. Although the TT01
/
I
genome contains Rhs-like elements [42], no Rhs element is
located in the genomic island encompassing the region H.
Nevertheless, we identified pairs of approximate long
repeated sequences (>1 kb) in direct orientation (RPT) that
corresponded to the observed deletion junction points.
Therefore, successive deletions in the region H are likely to
have been mediated by homologous recombination between
RPT during the transition from TT01
/I
to TT01α
/I
to VAR*,
leading to genomic reduction.

There was a strong selective pressure during the TT01α to
TT01α' lineage transition (3 months in LB broth without
shaking). This environmental constraint could thus be
responsible for the rearrangement leading to the region H
deletion. However, the region H deletion was already initi-
ated during the former transition (TT01
/I
to TT01α
/I
in the
laboratory-maintained nematode). Therefore, the observed
reduction genomic size is more likely to be the result of par-
ticular genomic features (the RPT) rather than environmental
constraints.
The region H is unique in the TT01
/I
genome. Nevertheless,
some RPT elements have similarities with sequences else-
where in the TT01
/I
genome, in the Photorhabdus strain W14
genome [55] or in other Enterobacteriaceae genomes such as
Yersinia pseudotuberculosis IP32953 (BX936398.1), Yers-
inia pestis Angola (CP000901.1), Yersinia pestis Pestoides F
(CP000668.1), Yersinia pestis CO92 (AL590842.1), Yersinia
pestis biovar Microtus str. 91001, (AE017042.1, Yersinia pes-
tis Antiqua (CP000308.1), Yersinia pestis Nepal 516
(CP000305.1), Yersinia pestis KIM (AE009952.1), Yersinia
pseudotuberculosis IP 31758 (CP000720.1), and E. coli
CFT073 (AE014075.1). Therefore, we propose that the region

H represents a new type of bacterial recombination hot spot,
which is vgr- and hcp-rich, but lacks Rhs elements.
A new duplication class
We described a single block duplication (region B) targeting a
275-kb region of the TT01
/I
genome in the TT01α' lineage.
This significant duplication encompasses 4.8% of the TT01
/I
genome. Region B is not located near the replication origin or
termination and does not correspond to genomic islands or
enterobacterial variable regions previously identified [42,56].
GC content or GC skew deviations are not evident.
Gene amplifications can occur through three kinds of known
mechanism: homologous recombination between direct
repeats, illegitimate recombination, or escape replication. No
repeated elements flanking region B were detected, despite
the use of the Nosferatu software [46], excluding the possibil-
ity of homologous recombination underlying this duplication.
Region B duplications may result from illegitimate recombi-
nation between short repeats [47,57,58]. However, amplifica-
tion copy number resulting from illegitimate recombination
events is often high, even for large amplicons, such as in Aci-
netobacter sp. ADP1 or Streptomyces kanamyceticus
[59,60]. Escape replication involves amplification of large
regions of the host genome (several hundred kilobases), next
to phage integration sites after induction of the phage lytic
cycle [61-64], or around degraded prophages without the
induction of specific phage lysis [65]. Although phage rem-
nants represent 4% of the Photorhabdus genome [42], lytic

phages have not been identified in Photorhabdus strains,
even after extensive investigation of lytic induction condi-
tions [66]. We detected the presence of an 11-kb phagic seg-
ment (plu0818-plu0826) in region B, potentially
representing a degraded prophage. However, whereas the
copy number usually resulting from the escape replication
mechanism ranges between three and ten, with its intensity
decreasing symmetrically from the center, region B in the
TT01α' lineage genomes represents a single block homogene-
ous duplication. We only identified one other previously
reported example of a large duplication without repeated
flanking sequences - a 250 kb duplication in Mycobacterium
smegmatis mc
2
155 genome [67]. Therefore, the duplication
of region B is likely to belong to a new class of duplications.
Observed phenotypes and global genomic architecture
are not systematically correlated
Large genomic changes such as deletions and duplications are
supposed to have important fitness effects. In our study, we
firstly demonstrated that the PV status (primary or second-
ary) is independent from global genomic architecture. This
was consistent with previous studies analyzing specific
genetic regions [28,38,39] and with partial genome studies
[33,40,41], but this is the first time it has been demonstrated
using a whole-genome approach.
We showed that the overall genomic pattern corresponds to
the variant lineage. Both the phenotype and pathogenic traits
of the primary PV (or the secondary PVs) are indistinguisha-
ble between the TT01 and TT01α lineages. Therefore, changes

in the genomic architecture of these strains did not lead to
observable changes in the phenotype. Furthermore, certain
regions that were deleted in the TT01α lineage potentially
encode biosynthesis pathways for antimicrobial compounds.
However, we did not observe any difference in antimicrobial
activity between TT01
/I
and TT01α
/I
. This finding suggests
that some TT01
/I
genes are redundant. Indeed, genes encod-
ing proteins potentially involved in the biosynthesis of anti-
microbial compounds are over-represented in TT01
/I
genome
[42]. Moreover, the encoded proteins in the deleted regions
may be adaptive factors required for specific conditions that
are not encountered in the laboratory or in our antibiosis
assays.
The TT01α' lineage differs from the two other lineages due to
its polymorphic genomic pattern. Furthermore, this lineage is
composed of three unstable CVs and the virulence of the
stabilized VAR* variant is attenuated in insects. This is con-
sistent with previously reports of CVs isolated from the Pho-
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.11
Genome Biology 2008, 9:R117
torhabdus genus [33]. Therefore, changes in genomic
architecture might be correlated to phenotypic changes in

variants of this lineage. The main rearrangement observed in
the TT01α' lineage is the region B duplication. According to
TT01
/I
genome annotation, region B may be involved in both
basal cellular functions and environment and/or host interac-
tions. Gene duplication events can underlie modification of
phenotypes [58]. However, we did not detect any modifica-
tion of gene transcription in region B using transcriptomic
microarray comparison between the VAR* and TT01α
/I
vari-
ants (Gaudriault S, unpublished data). Thus, this duplication
does not appear to modify gene expression in the VAR* vari-
ant. Therefore, the attenuation of virulence of the VAR* vari-
ant is not likely to be due to amplified expression in region B.
Rather, it is more likely that the 'cost' to the bacteria of the
increased genome size is decreased virulence in insects.
We conclude that the observed phenotypes and overall
genomic architecture are not systematically correlated in
TT01, TT01α, and TT01α' lineages. It is likely that this result
is general in the field of bacterial genomic architecture. Simi-
lar observations were previously made between strains of the
Pseudomonas aeruginosa species [68], but also inside a
clonal bacterial population of a wide range of bacterial groups
such as Yersinia pestis [19], Pseudomonas aeruginosa [17],
and Sinorhizobium meliloti [16].
Stability and plasticity of bacterial genome
architecture
Do large genomic rearrangements occur randomly or are they

shaped by drastic selective evolutionary forces? Several years
of comparative genomics between whole bacterial genomes
showed that the prokaryotic genome is a heterogeneous
entity, with regions of stability and flexibility [4,49,50].
Genomic stability is subject to selective pressures such as
functional replication [69], gene essentiality [70], or transla-
tion [71]. The three main routes of evolution of genome rep-
ertoire are lateral gene transfer, when several bacterial
communities share a same ecological niche, deletions, and
duplications [4,49,50]. The dynamism of genome repertoire
inside a clonal population only arises by the last two phenom-
ena, as illustrated by our study on Photorhabdus variants.
In E. coli, the chromosome is organized in structured macro-
domains, limiting genome plasticity. Whereas some genomic
rearrangements between these macrodomains have only
moderate effects on cell physiology, others have detrimental
effects [72]. The rearrangements that we observed in our var-
iants may have been selected to preserve chromosomal con-
figurations that are not detrimental for bacterial fitness in the
laboratory or in the nematode. We believe that structured
macrodomains that restrict chromosome plasticity are likely
to exist in other bacterial genus. Identification of structured
macrodomains in P. luminescens genome would provide bet-
ter knowledge on evolutionary forces modeling bacterial
genome.
Clonal variation, environmental adaptation, and
bacterial evolution
The major genomic variations described in TT01 variants
have cryptic consequences in our laboratory conditions. The
absence of associated phenotypes makes them difficult to

identify, explaining why such genomic variations are rarely
observed. However, further studies of such genomic varia-
tions may be crucial for a better understanding of bacterial
adaptation and evolution.
Indeed, we observed that the extensive genomic rearrange-
ments in Photorhabdus variants were often associated with
several genomic subpopulations in the same culture. Similar
observations were previously made for a P. luminescens
TT01
/I
locus encoding a phage tail-like structure [73] and the
mrf locus of the P. temperata strain K122 [74]. In Sinorhizo-
bium meliloti, Yersinia pestis, and Pseudomonas aerugi-
nosa, extensive variations of genome architecture, without
obvious changes in phenotype, were also observed during
bacterial growth in broth medium [16,17,19]. Different pre-
existing chromosomal forms in a clonal bacterial population
are likely to give this population an adaptive capacity. It is
therefore possible that bacterial populations maintain vari-
ous subpopulations with different genomic structures as a
way to cope with different environments during its life cycle.
Additionally, deletion events in TT01α and TT01α' lineages
are located within the TT01
/I
'flexible' gene pool. Whereas
intragenomic recombination in the 'flexible' gene pool have
been widely studied using comparative genomics for different
bacterial genera, species, and strains [1-4], similar reports for
clonal variants are rare. Gene repertoires of the 'flexible' gene
pool may evolve through variations in bacterial subpopula-

tions and then become fixed after bacterial speciation. Such
pre-existing or currently existing genomic variations have an
important role in evolutionary patterns of natural eukaryotic
populations [75]. They may also have a determinant role in
bacterial evolution.
Conclusion
The study of molecular mechanisms underlying genomic
plasticity in clonal populations is challenging because classi-
cal molecular tools only detect the major genomic state of the
population. Such studies are easier in bacterial species with a
high rate of bacterial variants. With our model, P. lumines-
cens, we identified two new genomic rearrangements, allow-
ing a new research axis for gaining a comprehensive
knowledge of bacterial chromosome plasticity. The cryptic
consequences of large genomic rearrangements in our model
also allow prospective comprehensive analysis of bacterial
genome evolution. Therefore, we propose that the P.
luminescens TT01 strain represents a new bacterial model for
study of genomic plasticity.
Genome Biology 2008, 9:R117
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.12
Materials and methods
Strains, plasmids, primers, and culture media
All bacterial strains and plasmids used in this study are listed
in Additional data file 6. Primers are listed in Additional data
file 7. P. luminescens was grown at 28°C in LB broth or on
nutrient agar 1.5% (BD Difco™, Franklin Lakes, New Jersey,
USA) for 48 hours. Escherichia coli was grown at 37°C in LB
broth or on LB supplemented with 1.5% agar (BD Difco™,
Franklin Lakes, New Jersey, USA). Strains were stored at -

80°C in LB broth containing 16% glycerol (vol/vol). Second-
ary variants were obtained by prolonged culture of primary
variants at 28°C for 10 days in Schneider's insect medium
(Cambrex Bio Science, Walkersville, Maryland, USA) with
shaking (TT01
/II
[30]), for 10 days in LB broth with shaking
(TT01α
/II
), or for 3 months in LB broth without shaking
(TT01α'
/II
). Secondary variant phenotypes were evaluated
from culture on NBTA (nutrient agar 1,5%, 25 mg/l bromoth-
ymol blue and 40 mg/l triphenyl-2,3,5-tetrazolium chloride)
plates and on TreGNO plates (see below) at 28°C. Secondary
variants were identified by performing phenotypic tests as
previously described [76] and controlled by PCR-restriction
fragment length polymorphism of the 16S rRNA gene [77].
Analysis of phenotypic variants on a new selective
medium: TreGNO
Xenorhabdus and Photorhabdus secondary variants are typ-
ically selected on NBTA plates to distinguish red secondary
variants colonies from blue primary colonies [76]. Because of
the high level of pigmentation of Photorhabdus colonies, the
use of color assays does not allow clear distinction between
primary and secondary variants for Photorhabdus genus. We
found that TT01 secondary variants were able to undergo tre-
halose fermentation, whereas primary variants can not. On
nutrient agar plates supplemented with trehalose (10 g/l) and

bromothymol blue (25 mg/l), secondary colonies acidified the
bromothymol blue and became yellow at 28°C after 48 hours.
Primary colonies remained green. Furthermore, secondary
colonies were flat and large with irregular borders. This new
medium was named TreGNO medium and was routinely used
for the discrimination of Photorhabdus luminescens strain
TT01 phenotypic variants.
PFGE and DNA electrophoresis
Intact genomic DNA was extracted in agarose plugs as fol-
lows. Bacterial cells grown on nutrient agar plates were sus-
pended in phosphate-buffered saline (GIBCO
®
Invitrogen,
Carlsbad, California, USA) to a turbidity of 1.25 at 650 nm,
included in 1% (vol/vol) low melting agarose (SeaPlaque
®
GTG, FMC BioProducts, Rockland, Massachusetts, USA)
solution and then subjected to lysis as described previously
[78].
NotI and ApaI hydrolysis were performed by incubation of
the agarose plugs overnight with 40 units of the endonuclease
in buffer recommended by the supplier (New England
Biolabs, Hertfordshire, UK), at 37°C for NotI and 25°C for
ApaI. PFGE was carried out in a contour-clamped homogene-
ous field electrophoresis apparatus CHEF-DRII (Bio-Rad,
Hercule, California, USA) in a 0.8% agarose gel in 0.5× Tris-
borate-EDTA (TBE) at 10°C. PFGE conditions were as fol-
lows: for NotI fragments, a 35 to 5 second pulse ramp for 47
hours followed by a constant pulse time of 50 seconds for 6
hours at 4.5 V/cm; and for ApaI fragments, 35 to 5 seconds

for 35 hours, followed by 5 seconds to 1 second for 4 hours at
4.5 V/cm.
I-CeuI hydrolysis was performed as described previously
[79]. For the separation of I-CeuI fragments, different elec-
trophoresis conditions were selected according to fragment
size: a pulse ramp from 5 to 50 seconds for 24 hours at 6 V/
cm for fragments with size below 700 kb; and a pulse ramp
from 150 to 400 seconds for 45 hours at 4.5 V/cm for I-CeuI
fragments for fragments between 700 kb and 1 megabase. For
I-CeuI fragments larger than 1 megabase, PFGE was per-
formed on Rotaphor apparatus (Biometra, Goettingen, Ger-
many) using 0.7% agarose gels in 0.5× TBE buffer. The
electrophoresis conditions used were as follows: 50 to 47 V
(linear ramp), 6,000 to 1,000 seconds decreasing pulses (log-
arithmic ramp), with a increasing angle from 96 to 105°,
buffer temperature 11°C, for 240 hours. I-CeuI PFGE patterns
were compared by calculating the Dice coefficient for each
pair [80]. Patterns were clustered by UPGMA using the
Phylip program package [81].
HindIII-hydrolyzed DNA was subjected to electrophoresis for
3 hours at 2.6 V/cm in a 0.8% agarose gel in 0.5× TBE using
SubCell apparatus (Bio-Rad) [13].
Southern blotting, probes, and hybridization
experiments
Electrophoresis gels were transferred onto a Nytran N Super-
Charge nylon membrane (Schleicher and Schuell, Dassel,
Germany) by vacuum blotting in 20 × SSC (Euromedex, Souf-
felweyersheim, France).
A digoxigenin-labeled probe targeting 16S rRNA gene was
obtained by PCR from genomic DNA of P. luminescens strain

TT01
/I
, using primers 27f and 1492r with a dNTP mixture
containing 0.1 mmol/l digoxigenin-dUTP [13].
Probes B and H were obtained using respectively small frag-
ment insert from plg2711 and large fragment inserts from
plbac4g08, plbac6h12, plbac3a10, plbac3c04, and plbac2f12.
Fragment inserts were purified, sonicated into fragments of
between 1 and 10 kb if insert size was higher than 10 kb, and
labeled with digoxygenin by random priming (Dig DNA labe-
ling Kit; Roche, Meylan, France). Hybridization of the probes
was detected using a CSPD chemiluminescent system
(Roche).
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.13
Genome Biology 2008, 9:R117
Standard DNA manipulations
Genomic DNA was extracted as previously described [56] and
stored at 4°C. We PCR-amplified the lopT1 deletion region
with Taq polymerase (Invitrogen, Carlsbad, California, USA),
in accordance with the manufacturer's recommendations,
using the PlopT1.fw an PlopT1.rev primers. The region H was
amplified by PCR with the Herculase Enhanced DNA
polymerase (Stratagene, Amsterdam Zuidoost, Pays Bas), in
accordance with the manufacturer's recommendations, using
the R-3236, F-3249, R-3238bis, and F-3254 primers. For
sequencing region H deletions, we purified the 4.8 kb and 5.2
kb fragments using the Montage PCR kit (Millipore, Guyan-
court, France) and sequenced using PCR primers and chro-
mosome walking (Millegen, Toulouse, France). Sequencing of
the 5.2 kb fragment central region of the fragment failed

probably because of the presence of repetitions. A 3.2 kb cen-
tral region was therefore amplified by PCR with PstIdMutF
and XbaIdMutR primers. The amplicon was hydrolyzed by
PstI and XbaI, ligated into PstI- and XbaI-hydrolyzed pUC19,
and inserted into E. coli XL1blue by transformation. The
resulting plasmid was purified by Nucleobond AX-100 kit
(Macherey-Nagel, Hoerd, France), and the insert was
sequenced with PstIdMutF and XbaIdMutR primers and
then by chromosome walking.
DNA microarray hybridization and analysis
DNA microarray hybridization and analysis were performed
as previously described [56].
Quantitative PCR analysis
Quantitative PCR was performed in triplicate using the Light-
Cycler FastStart DNA Master
PLUS
SYBR Green I kit from
Roche Diagnostics with 1 ng genomic DNA and 1 μmol/l spe-
cific primers targeting fliC (L-1954 and R-1954), mrfJ (L-
0778 and R-0778), dnaQ (L-0943 and R-0943), and pilN (L-
1051 and R-1051). The enzyme was activated for 10 minutes at
95°C. Reactions were performed in triplicate at 95°C for 5 sec-
onds, 60°C for 5 seconds and 72°C for 10 seconds (45 cycles),
and monitored in the Light Cycler (Roche). Melting curves
were analyzed for each reaction; all reactions exhibited a sin-
gle peak. The amount of PCR product was calculated with
standard curves obtained from PCR with serially diluted
TT01
/I
genomic DNA. All data are presented as ratios, with

gyrB (primers L-0004 and R-0004) as a control (95% confi-
dence limits).
Sequence analysis
Sequence annotation of the TT01
/I
genome was obtained from
the MaGe database [82]. We evaluated amino-acid and nucle-
otide similarity using BLASTP and BLASTN software [83].
We used Repseek software, previously Nosferatu [46], to
detect approximate repeats in large DNA sequences.
Pathogenicity assays
In vivo infection assays were performed as previously
described [45]. We performed three independent experi-
ments for each variant. Statistical analysis were performed as
previously described [84].
Antibiosis plate assays
Antibiosis assays were performed as previously described
[76] with the following bacterial species: Micrococcus luteus,
Staphylococcus epidermidis CIP 6821, Staphylococcus
aureus CIP 7625, Escherichia coli CIP 7624, Proteus vulgaris
CIP 5860, Pseudomonas aeruginosa CIP 76.110, Corynebac-
terium xerosis, Ochrobactrum intermedium LMG 3301
T
,
Ochrobactrum anthropi ATCC 49188
T
, Ochrobactrum sp.
FR49, Erwinia amylovora CFBP1430, Pseudomonas sp.
BW11M, Salmonella enterica 14028s, and Yersinia enteroco-
litica serotype 08.

Abbreviations
CV, colonial variant; kb, kilobase; NBTA, nutrient agar sup-
plemented with bromothymol blue and triphenyl-2,3,5-tetra-
zolium chloride; PCR, polymerase chain reaction; PFGE,
pulsed field gel electrophoresis; PV, phenotypic variant; Rhs,
recombination hotspot; RPT, repetition units; SCV, small-
colony variant; TBE, Tris-borate-EDTA; TreGNO, nutrient
agar with trehalose and bromothymol blue.
Authors' contributions
SG, SP, and AG characterized bacterial variants. SG, AL, and
CL provided molecular materials. SG and AL performed
microarray analysis. SG, CT, and EJ-B provided PFGE analy-
sis. SG analyzed sequence data. SG wrote the paper with con-
tributions from AG and EJ-B.
Additional data files
The following additional data files are available with this
paper. Additional data file 1 is a figure showing the deletion in
the lopT1 gene in TT01
/I
strain and the six variants.
Additional data file 2 is a figure showing PFGE of I-CeuI-
hydrolyzed genomic DNA of TT01
/I
strain and the six vari-
ants. Additional data file 3 is a figure showing the copy
number of 16S rDNA in TT01
/I
and the six variants. Addi-
tional data file 4 is a table listing the TT01
/I

missing genes in
TT01α
/I
and VAR* variants according whole-genome com-
parison using DNA microarray. Additional data file 5 is a
table listing the TT01
/I
amplified genes in TT01α
/I
and VAR*
variants, according to whole-genome comparison using DNA
microarray. Additional data file 6 is a table listing strains and
plasmids used in this study. Additional data file 7 is a table
listing primers used in this study.
Additional data file 1Deletion in the lopT1 gene in TT01
/I
strain and the six variantsPresented is a figure showing the deletion in the lopT1 gene in TT01
/I
strain and the six variants.Click here for fileAdditional data file 2PFGE of I-CeuI-hydrolyzed genomic DNA of TT01
/I
strain and the six variantsPresented is a figure showing PFGE of I-CeuI-hydrolyzed genomic DNA of TT01
/I
strain and the six variants.Click here for fileAdditional data file 3Copy number of 16S rDNA in TT01
/I
and the six variantsPresented is a figure showing the copy number of 16S rDNA in TT01
/I
and the six variants.Click here for fileAdditional data file 4TT01
/I
missing genes in TT01α
/I

and VAR* variantsPresented is a table listing the TT01
/I
missing genes in TT01α
/I
and VAR* variants according whole-genome comparison using DNA microarray.Click here for fileAdditional data file 5TT01
/I
amplified genes in TT01α
/I
and VAR* variantsPresented is a table listing the TT01
/I
amplified genes in TT01α
/I
and VAR* variants, according to whole-genome comparison using DNA microarray.Click here for fileAdditional data file 6Strains and plasmids used in this studyPresented is a table listing strains and plasmids used in this study.Click here for fileAdditional data file 7Primers used in this studyPresented is a table listing primers used in this study.Click here for file
Acknowledgements
This study received financial support from the Institut National de la
Recherche Agronomique (grant SPE 2004-1133-2). We thank Sylviane
Derzelle for the TT01
/II
gift, Eric Duchaud and Lionel Frangeul for help with
the circular map of DNA microarray data, Agnès Masnou and Emmanuelle
Genome Biology 2008, 9:R117
Genome Biology 2008, Volume 9, Issue 7, Article R117 Gaudriault et al. R117.14
d'Alençon for help in PFGE experiments, and Karine Brugirard-Ricaud for
lopT1 deletion identification. We thank Marie-Christine Guérin and Joël
Martin for expert technical assistance with quantitative PCR.
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